Alginate has been used to protect transplanted pancreatic islets from immune rejection and as a matrix to increase the insulin content of islet progenitor cells. The throughput of alginate bead generation by the standard extrusion and external gelation method is limited by the rate of droplet formation from nozzles. Alginate bead generation by emulsion and internal gelation is a scaleable alternative that has been used with biological molecules and microbial cells, but not mammalian cells. We describe the novel adaptation of this process to mammalian cell immobilization. After optimization, the emulsion process yielded 90 ± 2% mouse insulinoma 6 (MIN6) cell survival, similar to the extrusion process. The MIN6 cells expanded at the same rate in both bead types to form pseudo-islets with increased glucose stimulation index compared to cells in suspension. The emulsion process was suitable for primary pancreatic exocrine cell immobilization, leading to 67 ± 32 fold increased insulin expression after 10 days of immobilized culture. Due to the scaleability and broad availability of stirred mixers, the emulsion process represents an attractive option for laboratories that are not equipped with extrusion-based cell encapsulators, as well as for the production of immobilized or encapsulated cellular therapeutics on a clinical scale.

The long-term patency rates of vascular grafts and stents are limited by the lack of surface endothelialisation of the implanted materials. We have previously reported that GRGDS and WQPPRARI peptide micropatterns increase the endothelialisation of prosthetic materials in vitro. To investigate the mechanisms by which the peptide micropatterns affect endothelial cell adhesion and proliferation, a TAMRA fluorophore-tagged RGD peptide was designed. Live cell imaging revealed that the micropatterned surfaces led to directional cell spreading dependent on the location of the RGD-TAMRA spots. Focal adhesions formed within 3 h on the micropatterned surfaces near RGD-TAMRA spot edges, as expected for cell regions experiencing high tension. Similar levels of focal adhesion kinase phosphorylation were observed after 3 h on the micropatterned surfaces and on surfaces treated with RGD-TAMRA alone, suggesting that partial RGD surface coverage is sufficient to elicit integrin signaling. Lastly, endothelial cell expansion was achieved in serum-free conditions on gelatin-coated, RGD-TAMRA treated or micropatterned surfaces. These results show that these peptide micropatterns mainly impacted cell adhesion kinetics rather than cell proliferation. This insight will be useful for the optimization of micropatterning strategies to improve vascular biomaterials.

Additive manufacturing is now considered as a new paradigm that is foreseen to improve progress in many fields. The field of tissue engineering has been facing the need for tissue vascularization when producing thick tissues. The use of sugar glass as a fugitive ink to produce vascular networks through rapid casting may offer the key to vascularization of thick tissues produced by tissue engineering. Here, a 3D printer head capable of producing complex structures out of sugar glass is presented. This printer head uses a motorized heated syringe fitted with a custom made nozzle. The printer head was adapted to be mounted on a commercially available 3D printer. A mathematical model was derived to predict the diameter of the filaments based on the printer head feed rate and extrusion rate. Using a 1 mm diameter nozzle, the printer accurately produced filaments ranging from 0.3 mm to 3.2 mm in diameter. One of the main advantages of this manufacturing method is the self-supporting behaviour of sugar glass that allows the production of long, horizontal, curved, as well as overhanging filaments needed to produce complex vascular networks. Finally, to establish a proof of concept, polydimethylsiloxane was used as the gel matrix during the rapid casting to produce various "vascularized" constructs that were successfully perfused, which suggests that this new fabrication method can be used in a number of tissue engineering applications, including the vascularization of thick tissues.